Final Report Summary - V-RNA (Two facets of viral RNA: mechanistic studies of transcription and replication by influenza-like viral polymerases and detection by the innate immune system)
The RNA genome of viruses such as influenza is the centerpiece of molecular warfare between virus and host. A successful infection requires that the viral RNA is replicated and packaged into progeny virions, whereas to counter viral replication, host cells possess innate immune receptors that specifically recognize viral RNA as non-self thus triggering a powerful anti-viral response. This has led viruses to evolve counter-counter mechanisms to disrupt innate immune signaling pathways. The V-RNA project sought to elucidate some of the key molecular mechanisms involved in these processes using structural biology as the main tool.
A major objective of V-RNA was to provide a detailed picture of how the RNA-dependent RNA polymerase of influenza virus transcribes and replicates the single-stranded viral RNA (vRNA). This required first finding ways to produce milligram amounts of active recombinant polymerase, then crystallising the protein and determining its structure by X-ray crystallography. The obtained atomic resolution crystal structures (and most recently cryo electron microscopy structures) of the entire influenza polymerase were a breakthrough for the field. They show in detail how the polymerase binds to the vRNA promoter, which comprises both extremities of the vRNA, how the polymerase uses short capped primers to initiate transcription of viral mRNAs and how subsequent steps in transcription, including a special mechanism of self-polyadenylation, are likely to occur. The capped primers are pirated from host cell polymerase II (Pol II) transcripts by a unique process called ‘cap-snatching’, which is one mechanism by which the virus suppresses host cell gene expression. We structurally characterized the direct interaction between the phosphorylated C-terminal domain (CTD) of Pol II and influenza polymerase and showed that this is essential for viral transcription. Our detailed insights into influenza polymerase structure and mechanism have highlighted new opportunities to develop novel anti-influenza drugs and these have been followed up in spin-off projects in collaboration with other academic labs and pharmaceutical companies. In parallel, we solved the first structure of the RNA polymerase from a related segmented RNA virus from the bunyavirus family. This showed that despite high sequence divergence, the single chain bunyavirus polymerase (L protein) is architecturally and mechanistically similar to the heterotrimeric influenza polymerase.
The second major objective of V-RNA was to elucidate how in vertebrates, the RIG-I like family of cytosolic innate immune pattern recognition receptors (PRR) specifically recognize viral RNA, how this triggers a signaling pathway ultimately generating interferon expression and an anti-viral response and how certain viral proteins can disrupt this signaling pathway. In previous work we showed that the PRR RIG-I, an RNA helicase like molecule, switches conformation upon recognition and binding of a particular RNA structural motif, characteristic of viral RNA, thus releasing previously sequestered domains that initiate signaling. A critical next step is ubiquitination of these RIG-I signaling domains by the E3-ligase TRIM25, which promotes interaction of RIG-I with the downstream mitochondrial anti-viral signaling (MAVS) protein. We determined a crystal structure of TRIM25 showing where the RIG-I recognition domain of TRIM25 is positioned within the overall architecture of the molecule. Combining this result with the structure by a collaborator of the complex between TRIM25 and the influenza virus NS1 protein, reveals the mechanism by which NS1 inhibits RIG-I signaling, namely by competing with and displacing the RIG-I recognition domain of TRIM25 from its functionally active position. In the V-RNA project, we also worked on the two other RIG-I like helicases MDA5, which is activated upon detection of long dsRNA of viral origin, and LGP2, which binds RNA but lacks signaling domains. We determined RNA-bound crystal structures of MDA5 and LGP2 (the first of this PRR) and proposed a model of how LGP2, which is primarily a dsRNA end-binder like RIG-I, could promote MDA5 activation by nucleating MDA5 coating of dsRNA from one end. We also addressed the question of how the RIG-I like receptors avoid unwanted and deleterious activation by self-RNA that resembles viral RNA. We found that more weakly bound, near-cognate RNA is released by the ATPase activity of RIG-I or MDA5 before signaling can occur, consistent with the fact that certain genetic mutations which inhibit this ATPase activity can cause chronic inflammation due to autoimmunity. Finally, we solved high resolution structures of RIG-I bound to the influenza virus vRNA promoter, the first showing how RIG-I accommodates a dsRNA stem with non-canonical base pairs. Determination of the structures of both influenza polymerase and RIG-I bound to the same vRNA promoter symbolizes the success of the V-RNA project, which focused on viral RNA as the central protagonist in the molecular warfare between RNA viruses and their hosts.
A major objective of V-RNA was to provide a detailed picture of how the RNA-dependent RNA polymerase of influenza virus transcribes and replicates the single-stranded viral RNA (vRNA). This required first finding ways to produce milligram amounts of active recombinant polymerase, then crystallising the protein and determining its structure by X-ray crystallography. The obtained atomic resolution crystal structures (and most recently cryo electron microscopy structures) of the entire influenza polymerase were a breakthrough for the field. They show in detail how the polymerase binds to the vRNA promoter, which comprises both extremities of the vRNA, how the polymerase uses short capped primers to initiate transcription of viral mRNAs and how subsequent steps in transcription, including a special mechanism of self-polyadenylation, are likely to occur. The capped primers are pirated from host cell polymerase II (Pol II) transcripts by a unique process called ‘cap-snatching’, which is one mechanism by which the virus suppresses host cell gene expression. We structurally characterized the direct interaction between the phosphorylated C-terminal domain (CTD) of Pol II and influenza polymerase and showed that this is essential for viral transcription. Our detailed insights into influenza polymerase structure and mechanism have highlighted new opportunities to develop novel anti-influenza drugs and these have been followed up in spin-off projects in collaboration with other academic labs and pharmaceutical companies. In parallel, we solved the first structure of the RNA polymerase from a related segmented RNA virus from the bunyavirus family. This showed that despite high sequence divergence, the single chain bunyavirus polymerase (L protein) is architecturally and mechanistically similar to the heterotrimeric influenza polymerase.
The second major objective of V-RNA was to elucidate how in vertebrates, the RIG-I like family of cytosolic innate immune pattern recognition receptors (PRR) specifically recognize viral RNA, how this triggers a signaling pathway ultimately generating interferon expression and an anti-viral response and how certain viral proteins can disrupt this signaling pathway. In previous work we showed that the PRR RIG-I, an RNA helicase like molecule, switches conformation upon recognition and binding of a particular RNA structural motif, characteristic of viral RNA, thus releasing previously sequestered domains that initiate signaling. A critical next step is ubiquitination of these RIG-I signaling domains by the E3-ligase TRIM25, which promotes interaction of RIG-I with the downstream mitochondrial anti-viral signaling (MAVS) protein. We determined a crystal structure of TRIM25 showing where the RIG-I recognition domain of TRIM25 is positioned within the overall architecture of the molecule. Combining this result with the structure by a collaborator of the complex between TRIM25 and the influenza virus NS1 protein, reveals the mechanism by which NS1 inhibits RIG-I signaling, namely by competing with and displacing the RIG-I recognition domain of TRIM25 from its functionally active position. In the V-RNA project, we also worked on the two other RIG-I like helicases MDA5, which is activated upon detection of long dsRNA of viral origin, and LGP2, which binds RNA but lacks signaling domains. We determined RNA-bound crystal structures of MDA5 and LGP2 (the first of this PRR) and proposed a model of how LGP2, which is primarily a dsRNA end-binder like RIG-I, could promote MDA5 activation by nucleating MDA5 coating of dsRNA from one end. We also addressed the question of how the RIG-I like receptors avoid unwanted and deleterious activation by self-RNA that resembles viral RNA. We found that more weakly bound, near-cognate RNA is released by the ATPase activity of RIG-I or MDA5 before signaling can occur, consistent with the fact that certain genetic mutations which inhibit this ATPase activity can cause chronic inflammation due to autoimmunity. Finally, we solved high resolution structures of RIG-I bound to the influenza virus vRNA promoter, the first showing how RIG-I accommodates a dsRNA stem with non-canonical base pairs. Determination of the structures of both influenza polymerase and RIG-I bound to the same vRNA promoter symbolizes the success of the V-RNA project, which focused on viral RNA as the central protagonist in the molecular warfare between RNA viruses and their hosts.